The invention relates to a method for the analysis of chemical samples according to the preamble of claim 1. The invention also relates to a separation arrangement as used for example in high performance liquid chromatography (HPLC) according to the preamble of claim 4.
In the field of modern chemical analysis various chromatographic techniques are employed for their high separation efficiencies. Thus separation methods such as gas chromatography, microbore liquid chromatography, capillary supercritical fluid chromatography, capillary zone electrophoresis and high performance liquid chromatography are most commonly used. Associated with the separation systems are various kinds of different detectors. For example in high performance liquid chromatography the most popular detectors are the ones, which employ optical detection methods to measure changes in the absorption, in the luminescence, more particularly in the fluorescence, in the optical activity in general or in the refractive index of the carriers. As a major drawback the changes in absorption, in fluorescence or in optical activity, which are to be detected, are very often associated with changes of the refractive index of the carrier.
The change of the refractive index of the carrier results very often in a non-linear response of the detector, in an increased noise level or even in a spurious response of the detector. It is apparent that these effects are detrimental to the quality of the analysis of a chemical sample and in some cases render quantitative evaluations of chromatograms impossible. Aware of these drawbacks instrument-manufacturers have tried to solve these problems by improving the design of such separation systems and of the associated detector cells as outlined e.g. in an article by A. E. Bruno in Analytical Chemistry 1989, 61, pages 876 to 883. Now, while the approach of the instrument-manufacturers has had some success in reducing the influences of refractive index changes the problem still has not been satisfactorily resolved and to date no truly refractive index insensitive separation system has been designed. In their article Bruno et. al. also point out that the disturbing refractive index effects are partially a result of a complex dynamic distortion of the optical path, due for example to Schlieren effects, which make it even more difficult to obtain a proper correction of the distorted chromatograms.
It is therefore an object of the instant invention to avoid these drawbacks of the prior art separation methods and the respective prior art separation systems. In particular it is an object to eliminate the detrimental effects due to variations of the refractive index of the carrier during the separation.
All these and still further objects are achieved by a separation method comprising the steps outlined in the second part of claim 1 and by an analytic separation arrangement comprising the features claimed in claim 4.
The object is solved in particular by a separation method for the analysis of chemical samples, wherein a chemical sample is introduced into a carrier, which is transported through a system of tubes and past an optical detector, and together with the carrier is transported through a separation zone, preferably a separation column, and wherein after passing the separation column the carrier and the sample, which then is separated into its components, are transported past the optical detector, which is preferably constantly monitoring the carrier with respect to changes in absorption, in fluorescence or in optical activity. According to the invention prior to being transported past the optical detector the carrier, which is comming from the separation zone, is directed through a refractive index equalizing unit, where its refractive index is preferably constantly monitored and where upon detection of changes of the refractive index of the carrier the deviation is compensated by adding to the carrier a compensating agent.
In a preferred embodiment of the invention the carrier is transported inside the refractive index equalizing unit through a refractive index detector, which is connected with a controller unit. Upon detection of a deviation of the refractive index of the carrier, the controller unit controls the amount of compensating fluid, which is added to the carrier via a mixing chamber arranged upstream the refractive index detector, until the nominal value of the refractive index of the carrier is reached again.
The flexibility of the inventive method is increased even more, if depending on the detection of positive or negative refractive index deviations, a controlled amount of compensating agent having a refractive index lower or higher respectively than the refractive index of the carrier is added.
An analytic separation arrangement according to the invention comprises a system of tubes including a separation zone, preferably a separation column, which tubes upstream the separation zone are connected with reservoirs for a carrier and a chemical sample to be separated and analyzed, and downstream the separation zone are connected with a waste container for the carrier containing the sample and also are connected with transporting means for the carrier and the sample. The tube system is associated with an optical detector for monitoring changes in the absorption, in the fluorescence or in the optical activity of the carrier when it is transported through the tube system and past the optical detector, which is arranged in the path of the carrier behind the separation zone but in front of the waste container. Between the separation zone and the optical detector there is arranged in the path of the carrier a refractive index equalizing unit.
In a preferred embodiment according to the invention the refractive index equalizing unit comprises a refractive index detector, a mixing chamber, which is arranged upstream the refractive index detector in the path of the carrier and is connected to a reservoir for a compensating agent, and a controller unit, which on the one hand is connected with the refractive index detector and on the other hand with a dispensing device associated with the reservoir for the compensating agent.
The flexibility of the analytical separation arrangement according to the invention is considerably increased if the mixing chamber is connected with two reservoirs for two kinds of compensating agents, one having a refractive index higher than the nominal value of the refractive index of the carrier and the other having a refractive index lower respectively. Thus it is possible to compensate a wide range of deviations from the nominal value of the refractive index of the carrier, positive deviations as well as negative ones. Preferably the refractive indices of the compensating agents differ considerably from the nominal value of the refractive index of the carrier.
It is advantageous for the sensitivity and the accuracy of the analytical separation arrangement, when the total active volume of the refractive index equalizing unit, comprised of the volumes of carrier in the mixing chamber, the refractive index detector and the tubing interconnecting these two elements, is smaller than the volume of the optical detector.
In a preferred embodiment of the invention the total active volume of the equalizer amounts from about 0.5 .mu.l to about 1 ml, preferably to about 10 .mu.l. In even more miniaturized embodiments of the invention, for example in on-chip realizations, the active volume of the equalizer can amount to as little as about 1 nl.
It is particularly advantageous that the set-up of the inventive analytical separation arrangement can be simplified by using low rate syringe pumps having a pumping rate from about 1 .mu.l/min to about 100 .mu.l/min as the dispensing devices.
It is to be noted that the set-up of the invention is even more simplified by the fact that the controller unit is an off-the-shelf microcomputer-controlled DC-motor controller for the syringe pumps.